Establishing the molecular basis of synaptic transmission underlies a molecular understanding of learning, behavior, and memory. Such information will be essential for the design and improvement of therapeutic agents that regulate synaptic function for the treatment of psychological and neurological disorders. Neurotransmitter release into the synapse is mediated by vesicles that dock and fuse with the synaptic membrane, using molecular machinery common to all eukaryotic cells. The SNARE proteins present on vesicle and target membranes contain helical regions whose association is believed to promote membrane fusion. The interaction of SNAREs is therefore strictly regulated in order to prevent inappropriate fusion. In this proposal, a combination of biophysical and biochemical methods will be used to decipher how SNAREs interact with regulatory factors, and how they are recycled after fusion. 1. nSecl is a syntaxin la-binding protein required for vesicle trafficking and exocytosis. The specificity of the nSecl/syntaxin la interaction will be investigated by structure-based site-directed mutagenesis. The hypothesis that the release of syntaxin la from nSecl involves inter-domain movements of the latter will be tested by measuring the affinity and kinetics of mutants whose inter-domain interfaces have been altered by site-directed mutagenesis. The biochemical activity that releases nSecl from syntaxin will be isolated from brain extracts and characterized biochemically and structurally. 2. The structure of a binary complex between the synaptic membrane SNAREs syntaxin la and SNAP-25, which is believed to be an intermediate in the assembly of the SNARE complex, will be determined. The structures of highly conserved domains outside of the helical SNARE regions from the v-SNARE rSec22b and the t-SNARE syntaxin 6 will also be determined. 3.The ATPase NSF mediates disassembly of the SNARE complex after fusion, so that the components can be recycled for subsequent rounds of fusion. The conformational changes induced in NSF by ATP hydrolysis and release will be studied using a combination of small-angle x-ray scattering and crystallography.